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Neutral phytase PHYMJ11 and gene and application thereof

A phytase and neutral technology, applied in the field of genetic engineering, can solve the problems of increasing feed costs, phytate phosphorus cannot be effectively used by animals, and waste of phosphorus sources

Active Publication Date: 2009-06-17
WUHAN SUNHY BIOLOGICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, phosphorus in the form of phytate phosphorus is difficult to be utilized due to the lack of enzymes capable of decomposing phytic acid in monogastric animals, and its utilization rate is only 0-40%.
Phytate phosphorus cannot be effectively used by animals, which causes many problems in the feeding process. First, it causes waste of phosphorus sources. On the one hand, the phosphorus sources in the feed cannot be effectively used; , and additional inorganic phosphorus must be added to the feed, which increases the cost of feed
In actual production, when inorganic phosphorus is added, it often causes animal poisoning due to residual elements such as fluorine and heavy metals in inorganic phosphorus
Second, the formation of high-phosphorus feces will pollute the environment. About 85% of the phytate phosphorus in the feed will be directly excreted by animals. A large amount of phosphorus in the feces will seriously pollute the water and soil
Third, phytate phosphorus is also an anti-nutritional factor, which will combine with various metal ions Zn during the digestion and absorption process in the gastrointestinal tract of animals. 2+ , Ca 2+ 、Cu 2+ , Fe 2+ etc. and protein sequestration into corresponding insoluble complexes, thus reducing the effective utilization of these nutrients by animals
Currently commercially produced phytases for monogastric livestock and poultry basically have no enzymatic activity in a neutral pH environment, so they cannot be used as feed for carps
So far, there is no report of phytase with high enzyme activity in neutral environment

Method used

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  • Neutral phytase PHYMJ11 and gene and application thereof
  • Neutral phytase PHYMJ11 and gene and application thereof
  • Neutral phytase PHYMJ11 and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Screening and Characterization of Phytase-producing Agrobacterium (Pedobacter sp.MJ11)

[0077] Pedobacter sp.MJ11 is derived from the root soil of Saussurea chinensis in low temperature environment and has low temperature characteristics. Its genomic DNA was extracted, and determined by 16S rDNA sequence, it had the highest sequence similarity with Pedobacter sp.Ellin108, combined with morphological observation and physiological and biochemical characteristics, it was determined that it belonged to the genus Pedobacter. After activation with solid LB medium, transfer to low phosphorus medium (LPM, containing 1 g of casein peptone; 0.45 g of soybean peptone; 4 g of glucose; 0.5 g of glutamic acid; 5 g of sodium chloride; MgCl2 1.7 mM; MgSO4 1.4 mM ; KCl, 0.47mM; CaCl2 0.3mM; dissolved in 900mL 50mM pH7.5 Tris-HCl, dilute to 1000mL, aliquoted, sterilized at 15 lbs for 15min for use.), 30°C constant temperature rotary shaking culture for 24-36h. Take the supern...

Embodiment 2

[0078] Example 2 Cloning of the phytase-encoding gene phyMJ11 from Agrobacterium phytase

[0079] To extract the genomic DNA of Pedobacter sp.MJ11: take the bacterial solution cultured at 37°C for 2 days and centrifuge at 10000rpm for 10min. Take 100 mg of bacterial cells and add 500 μL of sterile water to wash, and centrifuge to get the precipitate. The precipitate was resuspended in 500 μL extract mixture, incubated at 37°C for 60 min, and centrifuged at 10,000 rpm for 10 min to remove the precipitate. The supernatant was extracted sequentially with equal volumes of phenol, phenol:chloroform, and chloroform. Take the upper layer solution and add 0.6-1 times the volume of isopropanol to precipitate at room temperature for 10 minutes. Centrifuge at 12000rpm for 15min. The precipitate was washed with 70% ethanol, centrifuged slightly, dried and dissolved in 30 μL sterile water for later use.

[0080] The degenerate primers were designed and synthesized according to the cons...

Embodiment 3

[0082] The activity analysis method of embodiment 3 phytase

[0083] The enzyme activity assay method is: the enzyme liquid is mixed with 0.1mol / L pH7.0 Tris-HCl containing 0.05% BSA and 0.05% Triton X-100 and added 1mM CaCl 2 Dilute with buffer solution, take 50μL diluted enzyme solution plus 950μL of substrate 1.5mmol / L sodium phytate (prepared with 0.1mol / L pH7.0 Tris-HCl buffer, and add 1mM CaCl 2 ), react at 37°C for 15 min, add 1 mL of 10% TCA to terminate the reaction, add 2 mL of chromogenic solution (10 g of ammonium molybdate tetrahydrate + 32 mL of sulfuric acid + 73.2 g of ferrous sulfate, add water to make up to 1 L). For the control, after adding enzyme solution, first add TCA and mix well, then add substrate. After color development, the OD value was measured at 700 nm, and the enzyme activity was calculated.

[0084] An enzyme activity unit (U) is defined as: under certain conditions, the amount of enzyme required to release 1 nmol of inorganic phosphorus per...

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Abstract

The invention relates to the genetic engineering field, especially to a land bacillus Pedobacter sp. MJ11 with a preservation number of CGMCC No. 2503 and a neutral phytase PHYMJ11, a gene thereof, a recombinant vector containing the same and its application. The invention provides the neutral phytase PHYMJ11 with an amino acid sequence shown as SEQ ID NO. 1 and a gene phyMJ11 for coding said neutral phytase. The neutral phytase obtained in the invention has the optimum pH value 7. 0 and simultaneously has a high enzymatic activity in a range of pH5-9. The ability for hydrolyzing a phytate phosphorus in a bean pulp of the phytase in a neutral condition is better than the present used phytase, which enables the phytase in the invention to apply to a fresh water fish feed stuff.

Description

technical field [0001] The present invention relates to the field of genetic engineering, specifically, the present invention relates to an Agrobacterium Pedobacter sp.MJ11, its preservation number is CGMCC No.2503, and the neutral phytase PHYMJ11 obtained therefrom and its gene, including the gene Recombinant vectors and applications. Background technique [0002] Phosphorus is an essential mineral that all animals need for growth. It is very important to provide adequate phosphorus in animal diets. Long-term phosphorus deficiency can lead to rickets in growing animals and osteoporosis in adult animals, thereby affecting the growth performance of animals. At present, inorganic phosphorus such as calcium hydrogen phosphate is added to animal feed to meet the animal's demand for phosphorus. However, animal diets that are mainly plant-based feeds are rich in phosphorus, but only because they mainly exist in the form of phytate phosphorus that animals cannot use. [0003] P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/16C12N15/56C12N15/63C12N1/21C12N1/19A23K1/165C12R1/01C12R1/19C12R1/645C12R1/07C12R1/225A23K20/189
Inventor 詹志春杨培龙邵娜黄火清王亚茹罗会颖柏映国
Owner WUHAN SUNHY BIOLOGICAL
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